The present invention relates to fluorescent structural analogs of the non-fluorescent nucleobases (amine bases, nitrogenous bases) and nucleosides commonly found in DNA and RNA, methods of their derivatization and subsequent use thereof use in the chemical or enzymatic synthesis of fluorescent oligonucleotides (polynucleotides), and to their new and useful application as probes in hybridization and sequencing reactions and the like. Additionally, the present invention relates to applications in which fluorescent structural analogs are substituted for specific non-fluorescent nucleosides in prescribed DNA or RNA sequences and to methods of using fluorescent oligonucleotides as hybridization reagents and probes for diagnostic and therapeutic purposes and as diagnostic and therapeutic research tools. More particularly, the present invention relates to maleimide derivatives of pyrimidines such as uridine and cytidine at their C-5 position, and to maleimide derivatives of purines such as adenine and guanine at their 8-position and to methods of their synthesis and the structure and synthesis of nucleoside tri-phosphates and phosphoramidites, etc., incorporating these derivatives.
The six commonly occurring N-nucleosides which predominate in the composition of DNA and RNA from all sources have the structures: 
wherein R6 is H for inosine and NH2 for guanosine, R9 is H for uridine and CH3 for thymidine. Furthermore, R12, R14=OH for ribonucleotides, R12=OH, R14=H for 2xe2x80x2-deoxy nucleotides, R12=H, R14=OH for 3xe2x80x2-deoxy nucleotides, and R12, R14=H in dideoxy nucleotides.
The six commonly occurring nucleotides do not absorb light at wavelengths greater than 290 nm and are effectively non-fluorescent under physiological conditions. Derivatives of the commonly occurring N-nucleotides for a variety of synthetic, diagnostic, and therapeutic purposes are common, including substitutions on both the heterocyclic base and the furanose ring. These substitutions can be made at the loci shown in: 
in which R4 is a reactive group derivatizible with a detectable label (NH2, SH,=O, and which can include an optional linking moiety including, but not limited to, an amide, thioether, or disulfide linkage or a combination thereof with additional variable reactive groups, R1 through R3, e.g., R1xe2x80x94(CH2)xxe2x80x94R2, or R1xe2x80x94R2xe2x80x94(CH2)x,xe2x80x94R3xe2x80x94, where x is an integer in the range of 1 and 25 inclusive; and R1, R2, and R3 can be a H, OH, alkyl, acyl, amide, thioether, or disulfide); R5 is H or part of an etheno linkage with R4; R6 is H, NH2, SH, or=O; R9 is hydrogen, methyl, bromine, fluorine, or iodine, or an alkyl or aromatic substituent, or an optional linking moiety including an amide, thioether, or disulfide linkage or a combination thereof such as R1xe2x80x94(CH2)xxe2x80x94R2, or R1xe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94, where x is an integer in the range of 1 and 25 inclusive; R10 is hydrogen, or an acid-sensitive base stable blocking group, or a phosphorous derivative, R11=R12=H; R12 is hydrogen, OH, or a phosphorous derivative; R is H, OH or OR where R is a protecting group or additional fluorophore. The letters N and C in the N-nucleosides and C-nucleosides designate the atom at which the glycosidic covalent bond connects the sugar and the heterocyclic base. In the cases of the commonly occurring nucleosides, the bases are either adenine, guanine, cytosine, inosine, uracil, or thymine. The bases are attached to a furanose sugar, a general structure of which is shown hereinunder. 
The sugar substituents for the fluorescent analogs share the same numbering system for all R groups, but the numbering system for some of the heterocycle analogs may differ.
Nucleotide sequences are commonly utilized in a variety of applications including diagnostic and therapeutic probes which hybridize target DNA and RNA and amplification of target sequences. It is often necessary, or useful, to label nucleotide sequences.
Labeling of oligonucleotide probes with radioisotopes. Hybridization of specific DNA or RNA sequences typically involves annealing oligonucleotides of lengths which range from as little as 5 bases to more than 10,000 bases (10 kb). The majority of oligonucleotide probes is currently in research use are radioactively labeled; however, because of (i) the short half lives of the isotopes in common usage, (ii) the safety requirements, and (iii) the costs of handling and disposal of radioactive probes, convenient and sensitive non-isotopic methods of detection are required for hybridization diagnostic methods to achieve widespread acceptance and application.
Non-isotopic methods of labeling oligonucleotide probes. In general, all of the non-isotopic methods of detecting hybridization probes that are is currently available depend on some type of derivatization of the nucleotides to allow for detection, whether through antibody binding, or enzymatic processing, or through the fluorescence or chemiluminescence of an attached xe2x80x9creporterxe2x80x9d molecule. In most cases, oligonucleotides have been derivatized to incorporate single or multiple molecules of the same reporter group, generally at specific cyclic or exocyclic positions.
Techniques for attaching reporter groups have largely relied upon (i) functionalization of 5xe2x80x2 or 3xe2x80x2 termini of either the monomeric nucleosides or the oligonucleotide strands by numerous chemical reactions using deprotected oligonucleotides in aqueous or largely aqueous media (see Cardullo et al. [1988] PNAS 85:8790-8794); (ii) synthesizing modified nucleosides containing (a) protected reactive groups, such as NH2, SH, CHO, or COOH, (b) activatable monofunctional linkers, such as NHS esters, aldehydes, or hydrazides, or (c) affinity binding groups, such as biotin, attached to either the heterocyclic base or the furanose moiety. Modifications have been made on intact oligonucleotides or to monomeric nucleosides which have subsequently been incorporated into oligonucleotides during chemical synthesis via terminal transferase or xe2x80x9cnick translationxe2x80x9d (see, e.g., Brumbaugh et al. [1988] PNAS 85:5610-5614; Sproat, B. S., A. L. Lamond, B. Beijer, P. Neuner, P. Ryder [1989] Nucl. Acids Res. 17:3371-3386; Allen, D. J., P. L. Darke, S J. Benkovic [1989] Biochemistry 28:4601-4607); (iii) use of suitably protected chemical moieties, which can be coupled at the 5xe2x80x2 terminus of protected oligonucleotides during chemical synthesis, e.g., 5xe2x80x2-aminohexyl-3xe2x80x2-O-phosphoramidite (Haralambidis, J., L Duncan, G. W. Tregar [1990] Nucl. Acids Res. 18:493-499); and, (iv) addition of functional groups on the sugar moiety or in the phosphodiester backbone of the polymer (see Conway, N. E., J. Fidanza, L. W. McLaughlin [19891 Nucl. Acids Res. Symposium Series 21:43-44; Agrawal, S., P. C Zamecnik [19901 Nucl. Acids Res. 18:5419-5423).
At the simplest, non-nucleoside linkers and labels have been attached to the 3xe2x80x2 or 5xe2x80x2 end of existing oligonucleotides by either enzymatic or chemical methods. Modification of nucleoside residues internal to the sequence of a DNA or RNA strand has proven to be a difficult procedure, since the reaction conditions must be mild enough to leave the RNA or DNA oligomers intact and still yield reaction products which can participate in normal Watson-Crick base pairing and stacking interactions.
Derivatizations of the heterocyclic base. Numerous methods for both cyclic and exocyclic derivatization of the N-nucleoside base have been described, including the following:
(i) Hapten labeling. DNA probes have been amino modified and subsequently derivatized to carry a hapten such as 2,4-dinitrophenol (DNP) to which enzyme-conjugated anti-hapten antibodies bind which subsequently can be processed using a calorimetric substrate as a label (Keller et al. [1988] Analytical Biochemistry 170:441-450).
(ii) Amino- and thiol-derivatized oligonucleotides. Takeda and lkeda ([1984] Nucl. Acids Research Symposium Series 15:101-104) used phosphotriester derivatives of putresceinyl thymidine for the preparation of amino-derived oligomers. Ruth and colleagues have described methods for synthesizing a deoxyuridine analog with a primary amine xe2x80x9clinker armxe2x80x9d 12 carbons in length at C5 (Jablonski et al. [1986] Nucl. Acids Res. 14:6115-6128). These were later reacted with fluorescein to produce a fluorescent molecule. Urdea and Horn were granted a patent in 1990 (U.S. Pat. No. 4,910,300) covering pyrimidine derivatives on which the 6-amino group at C4 had bee n modified. 3xe2x80x2 and 5xe2x80x2 amino modfying phosphoramidites have been widely used in chemical synthesis or derivatized oligonucleotides and are commercially available
(iii) Labeling with photobiotin and other biotinylating agents. The high affinity of biotin for avidin has been used to bind enzymatic or chemiluminescent reagents to derivatized DNA probes (Foster et al. [1985] Nucl. Acids Res. 13:745-761). Biotin conjugated to other linkers has also been widely used, including biotin-NHS esters (Bayer, E. A., M. Wilchek [1980] Methods in Biochemical Analysis 26:1), biotin succinamides (Lee, W. T., D. H. Conrad [1984] J Exp. Med. 159:1790), and biotin maleimides (Bayer, E. A. et al. [1985] Anal. Biochem 149:529). Reisfeld et al. ([1987] BBRC 142:519-526) used biotin hydrazide to label the 4-amino group of cytidine. A patent was granted to Klevan et al. in 1989 (U.S. Pat. No. 4,828,979) for such derivatizations at the 6-position of adenine, the 4-position of cytosine, and the 2-position of guanine. These derivatizations interfere with hydrogen bonding and base-pairing and have limited uses in producing oligomers for use in hybridization.
(iv) dU-Biotin labeling. Nucleoside 5xe2x80x2-triphosphates or 3xe2x80x2-O-phosphoramidites were modified with a biotin moiety conjugated to an aliphatic amino group at the 5-position of uracil (Langer et al. [1981] PNAS 78:6633-6637; Saiki et al. [1985] Science 230:1350-1354). The nucleotide triphosphate derivatives are effectively incorporated into double stranded DNA by standard techniques of xe2x80x9cnick translationxe2x80x9d. Once in an oligonucleotide, the residue may be bound by avidin, streptavidin, or anti-biotin antibody which can then be used for detection by fluorescence, chemiluminescence, or enzymatic processing.
(v) 11-digoxigenin-ddUTP labeling. The enzyme, terminal transferase, has been used to add a single digoxigenin-11-dideoxyUTP to the 3xe2x80x2 end of oligonucleotides. Following hybridization to target nucleic acids, DIG-ddUTP labeled hybridization probes were detected using anti-DIG antibody conjugate.
(vi) Immunofluorescent detection. Immunofluorescent detection can be done using monoclonal Fab"" fragments which are specific for RNA:DNA hybrids in which the probe has been derivatized with, e.g., biotin-11-UTP (Bobo et al. [1990] J. Clin. Microbiol. 28:1968-1973; Viscidi et al. [1986] J. Clin. Microbiol. 23:311-317).
(vii) Bisulfite modification of cytosine. Draper and Gold ([1980] Biochemistry 19:1774-1781) introduced aliphatic amino groups onto cytidine by a bisulfite catalysed termination reaction; the amino groups were subsequently labeled with a fluorescent tag. In this procedure, the amino group is attached directly to the pyramid base. Like the derivatization of uracil, these derivatizations interfere with hydrogen bonding and base-pairing and are not necessarily useful for producing efficient hybridization oligomers.
(viii) Fluorophore derivatized DNA probes. Texas Red (Sulfochloro-Rhodamine) derivatized probes are commercially available which hybridize to specific target DNAs and which can be detected using a flow cytometer or a microscope. Numerous authors have reported coupling fluorophores to chemically synthesized oligonucleotides which carried a 5xe2x80x2 or 3xe2x80x2 terminal amino or thiol group (Brumbaugh et al. [1988] Nucl. Acids Res. 16:4937-4956).
(ix) Direct enzyme labeling. Chemical coupling of an enzyme directly to a chemically synthesized probe has been used for direct detection through substrate processing. For example, Urdea et al. described an oligonucleotide sandwich assay in which multiple DNA probe hybridizations were used to bind target DNA to a solid phase after which it was further labeled with additional, alkaline phosphatase-derivatized hybridization probes (Urdea et al. [1989] Clin. Chem 35:1571-1575).
(x) Acridinium ester labeling. A single phenyl ester of methyl acridinium is attached at a central position on an RNA or DNA probe. Hydrolysis of the ester releases an acridone, CO2, and light. Because the ester on unhybridized probes hydrolyses more quickly than the ester on probes which have hybridized to target RNA or DNA, the chemiluminescence of the hybridized probes can be distinguished from that of free probes and is used in a xe2x80x9chybridization protection assayxe2x80x9d (Weeks et al. [1983] Clin. Chem. 29:1474-1479).
Derivatizations of the furanose ring. Methods for derivatization of the furanose ring (R11 through R14 hereinabove) and at the phosphodiester backbone of oligonucleotides (R10 hereinabove) have been reported.
(i) Internucleotide linkage reporter groups (R10 site). Phosphorothioate esters have been used to provide a binding site for fluorophores such as monobromobimane (Conway et al. [1989] Nucl. Acids Res. Symposium Series 21:43-44). Agrawal and Zamecnik ([1990] Nucl. Acids Res. 18:5419-5423) reported methods for incorporating amine specific reporter groups (e.g., monobromobimane) and thiol specific reporter groups (e.g., fluorescein isothiocyanate) through modifying the phosphodiester backbone of DNA to phosphoramidites and phosphorothioate diesters, receptively.
(ii) Glycosidic reporter groups R11 through R14 sites). Smith, Fung, and Kaiser ([1989] U.S. Pat. No. 4,849,513) described syntheses for an assortment of derivatives and labels on the glycosidic moiety of nucleosides and nucleoside analogs through the introduction of an aliphatic amino group at R10. The authors did not report or claim any uses or applications of inherently fluorescent oligonucleotides, either made chemically or enzymatically or using the fluorescent nucleoside analogs or their derivatives.
Fluorescent N-nucleosides and fluorescent structural analogs. Formycin A (generally referred to as Formycin), the prototypical fluorescent nucleoside analog, was originally isolated as an antitumor antibiotic from the culture filtrates of Nocardia interforma (Hori et al. [1966] J. Antibiotics, Ser. A 17:96-99) and its structure identified as 7-amino-3-b-D-ribofuranosyl (1H-pyrazolo-[4,3d] pyrimidine). This antibiotic, which has also been isolated from culture broths of Streptomyces lavendulae (Aizawa et al. [1965] Agr. Biol. Chem. 29:375-376), and Streptomyces gummaensis (Japanese Patent No. 10,928, issued in 1967 to Nippon Kayaku Co., Ltd.), is one of numerous microbial C-ribonucleoside analogs of the N-nucleosides commonly found in RNA from all sources. The other naturally-occurring C-ribonucleosides which have been isolated from microorganisms include formycin B (Koyama et al. [1996] Tetrahedron Lett. 597-602, Aizawa et al, supra; Umezawa et al. [1965] Antibiotics Ser. A 18:178-181), oxoformycin B (Ishizuka et al. [1968] J. Antibiotics 21:1-4; Sawa et al. [1968] Antibiotics 21:334-339), pseudouridine (Uematsu and Suahdolnik [1972] Biochemistry 11:4669-4674), showdomycin (Darnall et al. [1967] PNAS 57:548-553), pyrazomycin (Sweeny et al. [1973] Cancer Res. 33:26192623), and minimycin (Kusakabe et al. [1972] J. Antibiotics 25:44-47). Formycin, formycin B, and oxoformycin B are pyrazolopyrimidine nucleosides and are structural analogs of adenosine, inosine, and hypoxanthine, respectively; a pyrazopyrimidine structural analog of guanosine obtained from natural sources has not been reported in the literature. A thorough review of the biosynthesis of these compounds is available in Ochi et al. (1974) J. Antibiotics xxiv:909-916.
Physical properties of the N-nucleosides. Because several of the N-nucleosides were known to be active as antibiotic, antiviral, or anti-tumor compounds, their chemical derivatization and physical properties have been extensively studied and compared to the structures and syntheses of the N-nucleosides commonly found in DNA and RNA. In the late 1960s, several structural analogs of the six commonly occurring N-nucleosides were found to be fluorescent under physiological conditions; fluorescence in the analogs results from a molecular rigidity of the heterocycle structure itself; not all the structural analogs of a given type, e.g., the C-nucleosides, are fluorescent, nor is fluorescence an exclusive or inherent property of any particular class of structural analogs.
Nucleic acid hybridizations are now commonly used in genetic research, biomedical research and clinical diagnostics. In the basic nucleic acid hybridization assay, a single stranded nucleic acid (either deoxyribonucleic acid, DNA, or ribonucleic acid, RNA) is hybridized to a labeled nucleic acid probe and the resulting labeled duplexes are detected.
Chemical methods for incorporating modified nucleotides are described hereinabove and in PCT application WO 84/03285. The synthetic polynucleotides containing the modified nucleotides (usually referred as a xe2x80x9clinker arm nucleotidexe2x80x9d) can subsequently be derivatized with a fluorescent moiety. A review of labeling oligonucleotides with a variety of fluorescent molecules is described by Kessler in Nonradioactive Labeling and Detection of Biomolecules. Springer-Verlag Berlin Heidelberg New York, (1992).
So far, nucleosides were labeled using a linker arm. No work has been described in which increasing the fluorescence of, for example, uridine and cytidine is effected by extending the conjugation of the base moieties thereof by addition of an unsaturated moiety, as further detailed hereunder.
Fluorescent dyes have many uses and are known to be particularly suitable for biological applications in which the high detectability of fluorescence is desirable. For a discussion of the revolutionary impact fluorescent dyes have had, and will continue to have, on the way research is conducted today, please refer to Taylor et al. (1992) The New Vision of Light Microscopy, American Scientist, Vol. 80, pp. 322-335.
Ideally, improvement in detecting fluorescent probes in an assay system could be obtained by selecting a fluorophore which has (i) a large Stokes shift, that is, a large separation between the wavelengths for maximum excitation (EX) and the wavelength of maximal emission (EM), e.g., EM-EX greater than 100 nm); (ii) a high quantum yield (e.g., QY greater than 0.5); (iii) a high extinction coeffient (e.g., EC greater than 30,000); and (iv) an excitation maximum close to a laser line (e.g., 442 nm of Helium-Cadmium laser or 448 nm of Argon laser).
Unfortunately, there are no common fluorophores which fully satisfy these criteria. For example, fluorescein (EX: 495 nm, EM: 525 nm, QY=0.5) is a highly fluorescent label with an excitation maximum near a laser line, but has a Stokes shift of only about 30 nm.
It is known that a larger Stokes shift can be obtained by employing a pair of donor/acceptor fluorophores which have overlapping spectra and which are arranged in close proximity for non-radiative energy transfer between the donor and acceptor fluorophores.
This form of energy transfer was proposed by Forster, who developed equations of transfer efficiency in relation to separation distances between the fluorophores. See, for example, Forster, Th., Ann. Phys. (Leipzig) 2:55-75 (1948). A more recent summary of Forster""s non-radiative energy transfer is given in xe2x80x9cPrinciples of Fluorescent Spectroscopy,xe2x80x9d J. R. Lakowicz, Chapt. 10 (1983). The Forster mathematical analysis predicts that the closer the spacing of the fluorescent moieties the greater the efficiency of non-radiative energy transfer therebetween. Experimental evidence confirmed this prediction.
The use of multiple fluorescent dyes in a single measurement is ever growing, however, the number of distinguishable dyes is limited. It is for this reason that sophisticated staining techniques (combinatorial labeling or hybridization) and spectral imaging devices were required to enable distinctive fluorescent painting of the 24 human male chromosomes. See, E. Schroeck et al., 1996, Multicolor spectral karyotyping of human chromosomes. Science, 273, 494-497.
There is thus a widely recognized need for, and it would be highly advantageous to have, novel fluorescent nucleobases and nucleosides, which add to the ever increasing repertoire of fluorescent dyes.
The subject invention pertains to nucleobases and nucleoside structural analogs which are fluorescent. The fluorescent nucleoside structural analogs of the present invention are useful as monomers in synthesizing and labelling nucleoside sequences (oligonucleotides, polynucleotides). When used as hybridization probes, the fluorescence of such nucleoside sequences can be used as a research or diagnostic tool to detect and identify specific genetic sequences. This methodology is distinct from other non-radioactive methods of probe detection in that it does not utilize nucleosides which have been coupled to enzymes or other reactive proteins and does not require post-hybridization processing for the detection of hybridization. In their dideoxy form, the fluorescent nucleoside structural analogs of the present invention are useful as fluorescent chain elongation terminators in DNA sequencing reactions.
Thus, the nucleoside analogs according to the present invention can be used as (i) specific substitutes for a given non-fluorescent nucleoside in an oligonucleotide or polynucleotide probe, (ii) as labels for the identification and detection of specific sequences of a template; and (iii) for nucleic acid sequencing.
It is an object of the present invention to provide novel, inherently fluorescent nucleobases and nucleoside analogs and the novel triphosphate and phosphoramidite forms thereof, which are useful in the synthesis of labelled polynucleoside probes, amplimers, diagnostics, sequencing and therapeutics.
It is a further object of the present invention to provide methods of making autofluorescencing oligonucleotides and polynucleotides capable of specific Watson-Crick base pairing with prescribed sequences of target DNA or RNA.
It is another object of the invention to provide methods of using fluorescent nucleoside analogs and oligonucleotides made therefrom and synthesized according to the methods of the present invention to identify, detect the presence of, and/or alter the function of known nucleic acid sequences of DNA and RNA.
Thus, according to the present invention there is provided a compound of a general structure:
Dxe2x80x94Bxe2x80x94M
wherein:
B is selected from the group consisting of derivatives of naturally occurring nitrogenous bases having a Cxe2x80x94H group at positions 5 or 8, and derivatives of nitrogenous base-analogs having a Cxe2x80x94H group at positions 5 or 8;
D is at least one derivatizing group, including hydrogen; and
M is a maleimide derivative.
According to further features in preferred embodiments of the invention described below, B is selected from the group consisting of adenine derivative, guanine derivative, uracil derivative, cytosine derivative and inosine derivative.
According to still further features in the described preferred embodiments B is selected from the group consisting of purine derivative and pyrimidine derivative.
According to still further features in the described preferred embodiments D includes a chemical functionality group attached to a linker arm.
According to still further features in the described preferred embodiments D includes a ribose derivative.
According to still further features in the described preferred embodiments D further includes one to three phosphate groups attached at a 5xe2x80x2 position on the ribose derivative.
According to still further features in the described preferred embodiments D further includes a phosphoramidite derivative attached at a 3xe2x80x2 position on the ribose derivative.
According to still further features in the described preferred embodiments D includes a deoxyribose derivative.
According to still further features in the described preferred embodiments D further includes one to three phosphate groups attached at a 5xe2x80x2 position on the deoxyribose derivative.
According to still further features in the described preferred embodiments D further includes a phosphoramidite derivative attached at a 3xe2x80x2 or 5xe2x80x2 position on the deoxyribose derivative.
According to still further features in the described preferred embodiments D is a polymer.
According to still further features in the described preferred embodiments the polymer includes nucleoside derivatives.
According to still further features in the described preferred embodiments the polymer includes amino acid derivatives.
According to still further features in the described preferred embodiments the polymer is a polynucleotide.
According to still further features in the described preferred embodiments the polymer is a polypeptide.
According to still further features in the described preferred embodiments the polymer is a protein-nucleic acid polymer.
Further according to the present invention, there is provided a compound having a structure selected from the group consisting of: 
wherein:
R1, R2, R3, R4 and R5 are each independently a derivatizing group, including hydrogen.
According to still further features in the described preferred embodiments R1 is terminating with a reactive group having a general structure of:
(P)nA.
wherein:
P is selected from the group consisting of alkyl, branched alkyl, aromatic group, derivatized aromatic group and combinations thereof;
n is an integer in a range of 1 to 100; and
A is an active chemical moiety.
According to still further features in the described preferred embodiments n is an integer in a range of 2-50.
According to still further features in the described preferred embodiments n is an integer in a range of 3-7.
According to still further features in the described preferred embodiments A is capable of reacting with a nucleophile.
According to still further features in the described preferred embodiments the nucleophile is selected from the group consisting of amino and hydroxy groups.
According to still further features in the described preferred embodiments A is designed to form a chemical bond with the nucleophile, the bond is selected from the group consisting of ether bond, ester bond, amide bond, phosphonate bond, carbamate bond, and sulfone bond.
According to still further features in the described preferred embodiments A is an active ester.
According to still further features in the described preferred embodiments the active ester is selected from the group consisting of N-hydroxy succinimido, halogenoacyl, vinylsulfone, isothiocyanate, cyanate, chloromethyl ketone, iodoacetamidyl, iodoalkyl, bromoalkyl and active phosphate group.
According to still further features in the described preferred embodiments R2 and R3 are each independently selected from the group consisting of hydrogen, methyl, halogen, and an unsaturated moiety having a structure:
(CHxe2x95x90CH)mCH2Z
wherein:
m is an integer in a range of 1 to 6;
Z is selected from the group consisting of hydrogen, hydroxyl, amine, amide, nitro, an electron withdrawing group, an electron attracting groups and an aromatic group terminating with the hydrogen, hydroxyl, amine, amide, nitro, an electron attracting groups.
According to still further features in the described preferred embodiments R4 is selected from the group consisting of alkyl and aromatic group.
According to still further features in the described preferred embodiments R5 is selected from the group consisting of hydrogen and amino protecting group useful in a protection of amino acids in peptide synthesis.
According to still further features in the described preferred embodiments the amino protecting group is selected from the group consisting of trifluoroacetyl, acetyl, benzoyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 4-toluenesulfonylethyloxycarbonyl, methylsulfonylethyloxycarbonyl, 2-cyano-t-butyloxycarbonyl, chloroacetyl, acetoacetyl and 2,2,2-trichloroethyloxycarbonyl.
Further according to the present invention there is provided a compound having a structure selected from the group consisting of: 
wherein:
R2, R3, R4, R5, R6, R7, R8 and R12, are each independently a derivatizing group.
According to still further features in the described preferred embodiments R6 is a chemical functionality group.
According to still further features in the described preferred embodiments the chemical functionality group is selected from the group consisting of hydroxylic group OR9 and amino group NR10.
According to still further features in the described preferred embodiments R9 is an acid labile protecting group.
According to still further features in the described preferred embodiments the acid labile protecting group is selected from the group consisting of triphenylmethyl, p-anisyldiphenylmethyl, di-p-anisyldiphenylmethyl, p-dimethoxy trityltrityl, formyl, t-butyloxycarbonyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 4-chlorobenzoyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, furfurylcarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl, 2-phenylpropyl-(2)-oxycarbonyl, 2-(4-biphenyl)propyl-(2)-oxycarbonyl, 2-nitrophenylsulfenyl and diphenylphosphinyl.
According to still further features in the described preferred embodiments R9 is a base labile protecting group.
According to still further features in the described preferred embodiments the base labile protecting group is selected from the group consisting of trifluoroacetyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 4-toluenesulfonylethyloxycarbonyl, methylsulfonylethyloxycarbonyl, 2-cyano-t-butyloxycarbonyl, silyl ethers, and, 2,2,2-trichloroethylcarbonate.
According to still further features in the described preferred embodiments R10 is a nitrogen protecting group.
According to still further features in the described preferred embodiments the nitrogen protecting group is selected from the group consisting of trifluoroacetyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl 4-toluenesulfonylethyloxycarbonyl, methylsulfonylethyloxycarbonyl, and 2-cyano-t-butyloxycarbonyl, chloroacetyl, acetoacetyl, and 2,2,2-trichloroethyloxycarbonyl.
According to still further features in the described preferred embodiments R5 is a chemical functionality group.
According to still further features in the described preferred embodiments the chemical functionality group is an amino group NR10.
According to still further features in the described preferred embodiments R10 is an acid labile protecting group.
According to still further features in the described preferred embodiments the acid labile protecting group is selected from the group consisting of triphenylmethyl, p-anisyldiphenylmethyl, di-p-anisyldiphenylmethyl, p-dimethoxytrityl, formyl, t-butyloxycarbonyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 4-chlorobenzoyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, furfurylcarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl, 2-phenylpropyl-(2)-oxycarbonyl, 2-(4-biphenyl)propyl-(2)-oxycarbonyl, di-p-anisyldiphenylmethyl, 2-nitrophenylsulfenyl and diphenylphosphinyl.
According to still further features in the described preferred embodiments the base labile protecting group is selected from the group consisting of trifluoroacetyl, 9-fluorenylmethyloxycarbonyl, 4-toluenesulfonylethyloxycarbonyl, methylsulfonylethyloxycarbonyl and 2-cyano-t-butyloxycarbonyl.
According to still further features in the described preferred embodiments R7 is selected from the group consisting of hydrogen and OR11, wherein R11 is a chemical protecting group.
According to still further features in the described preferred embodiments the R11 protecting group is selected from the group consisting of lower aryl and alkylether.
According to still further features in the described preferred embodiments the R11 protecting group is selected from the group consisting of, triphenylmethyl, acetal, tetrahydropyranyl, silyl ether, trimethylsilyl and t-butyl-dimethylsilyl.
According to still further features in the described preferred embodiments the R11 protecting group is selected from the group consisting of hydroxylic and amino groups
According to still further features in the described preferred embodiments R8 is selected from the group consisting of lower and heterocyclic alkyl.
According to still further features in the described preferred embodiments R8 is selected from the group consisting of methyl, isopropyl, morpholino, pyrrolidino and 2,2,6,6-tetramethylpyrrolidino.
According to still further features in the described preferred embodiments R12 is a phosphate protecting group.
According to still further features in the described preferred embodiments the phosphate protecting group is selected from the group consisting of trichloroethyl, allyl, cyanoethyl and sulfonylethyl.
Further according to the present invention there is provided a compound selected from the group consisting of: 
wherein TPO is a triphosphate group, R2, R3 and R4 are each independently a derivatizing group, including hydrogen, and R7 and R13 are each independently selected from the group consisting of hydrogen and hydroxyl group.
According to still further features in the described preferred embodiments R2 and R3 are each independently selected from the group consisting of hydrogen, methyl, halogen, an unsaturated moiety having a structure:
(CHxe2x95x90CH)mCH2Z
wherein:
m is an integer in a range of 1 to 6;
Z is selected from the group consisting of hydrogen, hydroxyl, amine, amide, nitro, an electron withdrawing group, an electron attracting groups and an aromatic group.
According to still further features in the described preferred embodiments R4 is selected from the group consisting of alkyl and aromatic group.
Further according to the present invention there is provided a method of synthesizing a compound of a general structure:
Dxe2x80x94Bxe2x80x94M
wherein:
B is selected from the group consisting of derivatives of naturally occurring nitrogenous bases having a Cxe2x80x94H group at positions 5 or 8, and derivatives of nitrogenous base-analogs having a Cxe2x80x94H group at positions 5 or 8;
D is at least one derivatizing group, including hydrogen; and
M is a maleimide derivative;
The method comprising the steps of the method comprising the steps of contacting a derivatized base DB with mercuric acetate and condensing with N-alkylmaleimide.
Further according to the present invention there is provided a method of synthesizing a compound of a general structure: 
wherein:
R1, R2, R3, R4 and R5 are each independently a derivatizing group, including hydrogen; the method comprising the steps of contacting a derivatized base with mercuric acetate and condensing with N-alkylmaleimide.
Further according to the present invention there is provided a method of synthesizing a compound of a general structure: 
wherein R2, R3, R4, R5, R6, R7, R8 and R12, are each independently a derivatizing group;
The method comprising the steps of (a) contacting a nucleoside with mercuric salt, followed condensation with N-alkylmaleimide; (b) protecting amino groups of the nucleoside with a protecting group (e.g., allyloxycarbamate); (c) protecting 5xe2x80x2 hydroxyl of the nucleoside with an acid labile group (e.g., dimethoxytrityl); and (d) condensing with allylic phosphoramidate reagent.
Further according to the present invention there is provided a method of synthesizing a compound of a general structure: 
wherein TPO is a triphosphate group, R2, R3 and R4 are each independently a derivatizing group, including hydrogen, and R7 and R13 are each independently selected from the group consisting of hydrogen and hydroxyl group; the method comprising the steps of contacting a 5xe2x80x2 triphosphate nucleoside with mercuric salt and condensing with N-alkylmaleimide.
Further according to the present invention there is provided a method of hybridizing a target nucleic acid with a nucleic acid probe comprising the steps of contacting a sample including the target nucleic acid with the nucleic acid probe under hybridization conditions, wherein the nucleic acid probe includes at least one fluorescent derivative of a nucleobase, the fluorescent derivative includes a maleimide derivative attached at a C5 or C8 position of the nucleobase.
Further according to the present invention there is provided a method of detecting a sequence of target nucleic acid, comprising the steps of (a) providing the target nucleic acid in a single stranded form; (b) contacting, under hybridization conditions, the single stranded form of the target nucleic acid with a sequencing primer, such that a sequence dependent primer-target nucleic acid duplex is formed; and (c) contacting under polymerization conditions the duplex with deoxynucleoside-tri-phosphates and at least one dideoxynucleoside-tri-phosphate and with a DNA polymerase; wherein at least one of the sequencing primer, deoxynucleoside-tri-phosphates or the dideoxynucleoside-tri-phosphate includes a fluorescent derivative of a nucleobase, the fluorescent derivative includes a maleimide derivative attached at a C5 or C8 position of the nucleobase.
Further according to the present invention there is provided a method of synthesizing a polynucleotide comprising the steps of using a solid phase synthesis protocol to sequentially, following a predetermined order, add nucleoside derivatives present initially in their phosphoramidite form to a growing chain of the polynucleotide in a 3xe2x80x2 to 5xe2x80x2 direction, wherein at least one of the nucleoside derivatives includes a fluorescent derivative of a nucleobase, the fluorescent derivative includes a maleimide derivative attached at a C5 or C8 position of the nucleobase.
Further according to the present invention there is provided a method of synthesizing a polynucleotide comprising the steps of (a) providing the template nucleic acid in a single stranded form; (b) contacting, under hybridization conditions, the single stranded form of the template nucleic acid with at least one primer, such that a sequence dependent primer-target nucleic acid duplex is formed; and (c) contacting under polymerization conditions the duplex with deoxynucleoside-tri-phosphates and with a DNA polymerase; wherein at least one of the primers or the deoxynucleoside-tri-phosphates includes a fluorescent derivative of a nucleobase, the fluorescent derivative includes a maleimide derivative attached at a C5 or C8 position of the nucleobase.
Further according to the present invention there is provided a method of target dependent chemical ligation of probes comprising the steps of (a) providing a first probe including a fluorescence moiety including a is protected moiety being bound to the fluorescence moiety; (b) providing a second probe including a nucleophile moiety, the nucleophile moiety is selected such that when in appropriate proximity and orientation with respect to the protected moiety, the nucleophile moiety releases the protected moiety to yield a fluorescent moiety fluorescing or chemiluminescing differently than the fluorescence moiety; wherein the first and second probes are selected such that by hybridizing to the target the appropriate proximity and orientation are obtained.
The present invention successfully addresses the shortcomings of the presently known configurations by providing novel nucleobases and nucleoside structural analogs featuring improved spectral qualities by conjugating a maleimide derivative at C5 or C8 positions of pyrimidine nucleobases or purine nucleobases, respectively.